In recent years, it has been revealed that nitrogen monoxide (NO) is an endogenous physiologically active substance having various functions responsible for, for example, blood vessel relaxation, regulation of nervous signal transduction, control of cell death, carcinogenesis, and the like. It is considered that nitrogen monoxide itself has relatively weak reactivity, and is converted to various reactive nitrogen species (RNS) having high reactivity by reactions with various active oxygen species simultaneously produced in living bodies, metal ions and the like to cause cell injury. More recently, there are many reports teaching that various signal transduction routes are regulated by modification of proteins with RNS, and it is also being revealed that RNS including peroxynitrite participate in various diseases by giving the DNAs damage. Therefore, not only nitrogen monoxide but RNS have been attracting a great deal of attention.
Peroxynitrite (ONOO−) is a typical substance among RNS, and is produced by a reaction of nitrogen monoxide and superoxide. Reaction rate of this production reaction is mostly limited by diffusion, and when superoxide produced by NADPH oxidase or the like and nitrogen monoxide produced by nitrogen monoxide synthetase (NOS) coexist, peroxynitrite is immediately produced. Peroxynitrite has high oxidation ability, for example, it achieves nitration of an aromatic ring, and has characteristic reactivities such as, for example, efficient nitration of tyrosine. Recent reports have pointed out that phosphorylation of tyrosine is inhibited by nitration of tyrosine, and thus peroxynitrite has an important effect on signal transduction systems such as MAPK and PI3K/Akt cascades.
Examples of the methods for detecting peroxynitrite developed so far include (1) a method of detecting 8-nitroguanine produced by nitration of guanine which is a DNA base, or nitrotyrosine produced by nitration of tyrosine by HPLC or immunostaining using an antibody, and (2) a method of detecting singlet oxygen produced by reaction of peroxynitrite and hydrogen peroxide on the basis of light emission at 1.3 μm. Although the method (1) achieves high specificity and has been widely used, the method has a problem in that peroxynitrite cannot be detected in real time by applying the method to a living cell system, because HPLC analysis or staining with antibodies should be performed. In addition to the aforementioned two methods, (3) a chemiluminescence method using luminol, and (4) a fluorometric detection method using a fluorescence probe to detect overall active oxygen species such as 2′,7′-dichlorodihydrofluorescein (DCFH) have been used. However, these methods fail to achieve specificity, and therefore reliable detection cannot be expected even if various inhibitors are used. For example, in the method (4), DCFH reacts with both of nitrogen monoxide and superoxide to give an increase in fluorescence, and therefore it is impossible to distinguish whether peroxynitrite is detected, or nitrogen monoxide or superoxide is detected.
Arylated fluorescein derivatives are known to be useful fluorescent probes for measuring active oxygen (International Patent Publication WO01/64664). Moreover, these fluorescein derivatives are known to be useful as fluorescent probes which do not react with nitrogen monoxide and superoxide which are precursors of peroxynitrite, and thus enables measurement of peroxynitrite while distinguishing it from those precursors (International Patent Publication WO2004/40296). However, these fluorescein derivatives have a problem that they also react with reactive oxygen species (ROS) such as hypochlorite ion and hydroxyl radical, and thus are not capable of achieving specific detection solely of peroxynitrite while distinguishing it from other ROS. Therefore, it has been desired to develop a fluorescent probe which can highly selectively visualize peroxynitrite in a living cell or tissue.